The serum albumins are the major soluble proteins of the circulatory system and contribute to many vital physiological processes. Serum albumin generally comprises about 50% of the total blood component by dry weight, and as such is responsible for roughly 80% of the maintenance of colloid osmotic blood pressure and is chiefly responsible for controlling the physiological pH of blood. The albumins also play an extremely important role in the transport, distribution and metabolism of many endogenous and exogenous ligands in the human body, including a variety of chemically diverse molecules including fatty acids, amino acids, steroids, calcium, metals such as copper and zinc, and various pharmaceutical agents. The albumins are generally thought to facilitate transfer many of these ligands across organ-circulatory interfaces such as the liver, intestines, kidneys and the brain, and studies have suggested the existence of an albumin cell surface receptor. See, e.g., Schnitzer et al., P.N.A.S. 85:6773 (1988). The albumins are thus intimately involved in a wide range of circulatory and metabolic functions.
Human serum albumin (HSA) is a protein of about 66,500 kD protein and is comprised of 585 amino acids including at least 17 disulfide bridges. As with many of the albumins, human serum albumin plays an extremely important role in human physiology and is located in virtually every human tissue and bodily secretion. As indicated above, HSA has an outstanding ability to bind and transport and immense spectrum of ligands throughout the circulatory system including the long-chain fatty acids which are otherwise insoluble in circulating plasma. Certain details regarding the atomic structure and the binding affinities of albumin and the specific regions primarily responsible for those binding properties have previously been disclosed, e.g., in U.S. patent application Ser. No. 08/448,196, filed May 25, 1993, now U.S. Pat. No. 5,780,594 and U.S. patent application Ser. No. 08/984,176, filed Dec. 3, 1997, now U.S. Pat. No. 5,948,609, both of which are incorporated herein by reference.
Because of the vital role played by albumins, there are literally thousands of applications for serum albumin covering a wide range of physiological conditions. However, unlike blood proteins such as hemoglobin, native serum albumins are non-functional as oxygen transport systems, and thus have not been useful in blood replacement systems requiring oxygen transport. Accordingly, one recent focus of research has been the binding of the albumin molecule with heme, one of the important blood proteins. Under normal physiological conditions, heme that finds its way into plasma is bound by the specific heme-binding protein, hemopexin, which delivers it to the liver for excretion via a receptor-mediated uptake mechanism (1–5). Under pathophysiological conditions of severe hemolysis when significant amounts of free hemoglobin appear in the circulation, serum albumin can also become a significant transporter of heme (6,7), principally as hemin (FeIII Protoporphyrin-IX (Cl)). These are conditions when hemopexin becomes saturated by hemin, and albumin, which is present at considerably higher concentration than hemopexin, acts as a depot for the overflow. Additionally, a source of heme uptake by albumin has been suggested to result from the uptake of soluble heme-containing peptides released by the enzymatic digestion of dietary heme-containing proteins such as cytochrome c, where they may constitute a significant route by which iron enters the mammalian system (8).
Hemin is one of the important endogenous ligands transported and/or sequestered by human albumin and among the most highly bound having with a predicted a single high affinity site with KA=1.1×108 M−1 (9). Interestingly, among mammals, only albumin of primates shows a single high affinity heme binding site (4). Studies of heme binding to albumin suggest a two step binding process, a fast interaction to form an intermediate complex, followed by “internalization” of the hemin in a region with limited access to bulk aqueous solvent (9,10). Although various hypotheses concerning the binding location and chemistry to human albumin have been proposed from spectroscopic and other methods (11–17), except for the general binding location within cleavage fragments (IB-IIA) (6) and more recently recombinant domains (domain I) (18), the conclusions of all of the other studies are inconsistent with the location and coordination of the atomic structure of the complex in accordance with this invention as described herein.
Previously, the structure of human methemalbumin was determined at 2.8 Å (19) using a crystal form of the space group C2, i.e. form-III as reported in reference (20). These publications evidenced that the heme binds to albumin within the IB pocket, a site that was previously identified with long chain fatty acid transport. In addition, a successful blood replacement product was obtained which featured a heme-albumin complex with a recombinant serum albumin having at least one of the four key hydrophobic binding residues in the heme binding region replaced with a histidine, such as disclosed in U.S. Pat. No. 5,948,609, incorporated herein by reference. However, the ability to uncover additional information which would lead to further breakthroughs with regard to the capacity of albumin to be utilized to further improve gas binding properties has heretofore been limited by the resolution of the crystal albumin structure obtainable. Similarly, it is also important to obtain additional insights with regard to the heme molecule and its derivatives so as to be able to develop heme molecules and derivatives which when combined with albumin will provide oxygen or other gas binding/transport delivery properties or allow for other applications such as scavenging of toxic gases (e.g., cyanide, nitric acid, carbon monoxide, etc.).
Accordingly, it is highly desirable to develop a system of determining key binding regions of the heme/albumin complex and to develop a means by which the alteration of albumin or heme genetically can be accomplished so as to identify and maximize the medically relevant gas binding properties of these molecules, a goal that has not previously been achievable with lower resolution pictures of the albumin crystal complex structure at the relevant gas binding sites.